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Title: Lasso Peptide TonB-dependent Transporter: Structure, Mechanism and Phylogeny
Authors: Wangsanuwat, Chatarin
Advisors: Link, A. James
Department: Chemical and Biological Engineering
Class Year: 2016
Abstract: Lasso peptides are ribosomally-synthesized and post-translationally modified peptides with threaded lasso structures. Astexins-2 and -3 are 24- residue lasso peptides found in Asticcacaulis excentricus. Astexins-2 and -3 are hypothesized to be metal scavengers for the bacterial cells. Within lasso peptide gene clusters, there is a lesser-known gene and protein, hypothesized to be an outer membrane TonB-dependent transporter based on its homology. This set of proteins, named F-proteins, are predicted to transport lasso peptides acting as metal scavengers. The transport mechanism of the TonB-dependent transporter is still partly unclear, especially the function of the N-terminal extension found in some of the transporters. The project shows that an F-protein, AtxF2, whose gene lies within the same cluster as the genes coding for astexins-2 and -3, is targeted correctly to the outer membrane. Moreover, the project illustrated that E. coli, a gamma proteobacteria, could not successfully utilize AtxF2 originally from A. excentricus, an alpha proteobacteria, suggesting a class divergence among proteobacteria. In addition, initial findings suggest that metal ions positively affect astexin-3 transport in E. coli, and the bacterial cells transport astexins-2 and -3 together in a different way from transporting either peptide by itself, suggesting there might be some interaction between the two lasso peptides during the transport. A majority of F-proteins was found to possess an N-terminal extension. More than 90% of gene clusters possessing N-terminal extension F-proteins have a fecR-like gene upstream of the f-gene. These findings support the hypothesis that N-terminal extension of the transporter interacts with anti-σ factor FecR, and such interaction exposes a TonB-box, buried in the β-barrel, such that it can interact with TonB. Absence of E. coli’s TonB-box and conserved motifs in the transporters and their FecR-like proteins also indicate evolutionary divergence between proteobacteria classes. Phylogenetic trees construction of F-proteins suggests that f-gene has evolved along with the rest of the genome. Additionally, concatenated conserved motifs of F-proteins that are located in the vicinity can be used as an input to provide a similar phylogenetic tree as using the entire sequence as an input. The result suggests that only a conserved portion of the sequences is sufficient to provide an accurate estimation of the true phylogenetic tree, sparing time and computational resources. Moreover, two common phylogeny construction methods: posterior probability and maximum likelihood yield similar trees. The results of this project contribute to our understanding of F-proteins’ evolution and structure, with direct implication on the mechanism. These findings will aid in the development of TBDTs transport mechanism theory.
Extent: 72 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Chemical and Biological Engineering, 1931-2017

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